Apparatus and method for spotting a substrate

ABSTRACT

The present invention provides a method and apparatus for dispensing a small volume of a selected liquid, such as a biological sample or reagent, onto a substrate. The device includes a tube adapted to contain the liquid. An elongate fiber is disposed within the tube for axial movement therein between raised and lowered positions. Upon shifting or oscillating the fiber between its raised and lowered positions, a liquid spot can be formed at a selected position on the substrate. The device is readily adaptable for the production of micro-arrays having a great number of individual spots.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/270,218,filed Mar. 15, 1999 U.S. Pat. No. 6,296,702, which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to the dispensing of liquids. Moreparticularly, the invention provides an apparatus and method of use forspotting liquids, such as biological samples or reagents, onto asubstrate.

BACKGROUND OF THE INVENTION

Target compounds, or analytes, present within a sample can often beidentified through the controlled exposure of the sample to anappropriate probe, with subsequent detection of a resulting reaction. Ina typical arrangement, a sample of a test solution containing an analyteof interest is exposed to a probe carrying a detectable reporter. Theprobe is chosen such that it can specifically bind the analyte, e.g., byhybridization of complementary nucleotide sequences, or antibody-antigeninteractions. After excess probe material has been removed, e.g., washedaway, specific binding of the probe to the analyte can be detected.

As the sensitivity of analytical techniques continues to improve, it isincreasingly desirable to carry out such analyses using very smallvolumes of samples/reagents. This is especially true in situationsinvolving expensive compounds. Accordingly, it is now popular to utilizevery small volumes of such liquids laid down as “spots” on the surfaceof a substrate, such as a slide, microcard, or chip.

Not only is it often desirable to provide ultra-small volumes ofindividual samples and/or reagents in the form of spots, it is becomingincreasingly popular to arrange numerous such spots in close proximityto one another as an array on a substrate. For example, a lab technicianmight need to evaluate a specimen for the presence of a wide assortmentof target biological and/or chemical compounds, or to determine thereaction of many different specimens against one or more reagents, suchas labeled probes. High-density array formats permit many reactions tobe carried out in a substantially simultaneous fashion, saving space,time and money.

Both manual and automated devices for dispensing very small fluidvolumes have been devised, including, for example, micropipettes, pins,quills and ink-jetting devices. While suitable for some purposes, eachof these is associated with certain disadvantages. For example,micropipettes are generally incapable of accurately dispensing theextremely small volumes of liquid called for by many present-dayprotocols. With regard to pens and quills, a number of problems need tobe resolved relating to the differences in size and shape of the spotswhich are placed (which can lead to differences in resulting signalintensity or overlap of spots), “missed spots” (where little or nosample is placed on the surface), and the overhead associated withcleaning and reloading. Ink-jet devices dispense a controlled volume ofliquid onto a substrate by use of a pressure wave created within thecartridge. This approach is not acceptable for the spotting of samplescontaining relatively fragile macromolecules, as they can become sheeredor otherwise damaged. Further, ink-jetting devices are associated with ahigh degree of splattering, thereby presenting a substantial risk ofcontamination, particularly for closely spaced spots.

As an additional disadvantage, most of the known spotting devicesrequire very precise placement of the spotting head relative to thesubstrate surface. Variations in the distance between the spotting headand the substrate surface can result in inconsistent spot sizes and/ormissed spots. With particular regard to contact-type devices, if placedto close to the substrate, the spotting tip can collide with thesubstrate surface with a force sufficient to damage the spotting tipand/or the substrate.

In view of the above, the need is apparent for a device and methoduseful for delivering a micro-volume of liquid onto a substrate in aquick and precise manner. Preferably, the device should be relativelyeasy to use, cost effective and readily adaptable for the production ofmicro-arrays having a great number of individual spots.

SUMMARY OF THE INVENTION

In one of its aspects, the present invention provides an apparatus formicro-spotting a predetermined volume of a liquid at a plurality ofspaced regions on a substrate or substrates.

In one embodiment, the apparatus includes a tube adapted to contain aselected liquid, such as a biological sample, reagent, or the like. Thelower end of the tube defines an orifice having a diameter of less thanabout 1 mm. In an exemplary construction, the diameter of the orifice isless than about 500 μm, and preferably less than about 200 μm. Anelongate fiber is disposed within the tube for axial movement thereinbetween raised and lowered positions. The fiber, which has a free distalend, is provided with a diameter that is less than the inner diameter ofthe tube's lower end. In one exemplary arrangement, the diameter of thefiber is between about 10-100 μm smaller than that of the orifice. Forexample, a fiber having a diameter of about 100 μm can be disposedwithin a tube having an inner diameter of between about 110 to 200 μm. Aworkpiece holder can be employed to hold a selected substrate orsubstrates for spotting. At its raised position, the fiber's free end isspaced from the surface of such a substrate. At its lowered position,the fiber's free end contacts the surface of the substrate.

Shifting means are operatively connected to the fiber for shifting thesame between its raised and lowered positions. The shifting means canbe, for example, an actuator, such as a linear or vertical actuator, orthe like. Positioning means are provided for positioning the tube andassociated fiber laterally with respect to the workpiece holder, atselected deposition positions with respect to the substrate. Thepositioning means can be adapted to move the substrate and/or the tube,fiber and shifting means. In one embodiment, for example, thepositioning means is an x-y positioner (e.g., a robotically controlledx-y movable arm) operatively connected to the tube and the shiftingmeans.

A control unit is operatively connected to the positioning means andshifting means for use in successively (i) positioning the tube andassociated fiber at a selected deposition position with respect to asubstrate, and (i) shifting the fiber to its lowered position, todeposit a selected volume of liquid upon such substrate. In a preferredembodiment, the fiber is (i) laterally flexible and (ii) substantiallyincompressible along its longitudinal axis. Suitable fibers having suchcharacteristics include, for example, optical fibers. Advantageously,these characteristics permit efficient transfer of motion from theshifting means to the fiber, and the accommodation of variations in thedistance between the tube's lower end and the substrate by flexing, orbowing, of the fiber.

According to one embodiment, the apparatus is adapted for use inmicro-spotting a predetermined volume of a liquid at a preselectedposition on each of a plurality of different substrates in the workpieceholder. The control unit, in this embodiment, is operable to positionthe tube successively at such preselected position on each substrate.

In one embodiment, the tube has a substantially uniform diameter, andincludes a larger-diameter upper reservoir for holding the selectedliquid. The tube and reservoir can be separately formed and subsequentlyattached together, or they can be integrally formed.

In another embodiment, the tube's inner diameter tapers on progressingdownwardly to a defined-volume tube end region having the diameter ofthe orifice. The diameter of the tube end region, in this embodiment, issubstantially the same as that of the fiber. The fiber's end, with suchin its raised position, is disposed above the tube end region, such thatshifting of the fiber from its raised to its lowered position iseffective to expel from the tube the volume of liquid contained in thetube end region.

One embodiment of the apparatus, particularly useful in micro-spotting apredetermined volume of one or more selected liquids simultaneously atselected deposition regions on a substrate, includes a plurality oftubes, and associated fibers, and shifting means. The tubes can take theform, for example, of channels provided in a manifold. Fiber flexingaccommodates variations in the distance between the tubes' lower endsand the associated positions at the substrate.

The present invention further provides an apparatus for micro-spotting apredetermined volume of a selected liquid on a substrate, including atube having an inner diameter that tapers on progressing downwardly to adefined-volume tube end region having a substantially uniform diameterof less than about 1 mm. According to one embodiment, the diameter alongthe tube end region is less than about 500 μm, and preferably less thanabout 200 μm. The tube is adapted to contain the selected liquid bycapillary or surface tension forces. An elongate fiber having a diametersubstantially the same as that of the tube end region is disposed withinthe tube for axial movement therein between raised and lowered positionsat which the fiber's free end is disposed above and below the tube endregion, respectively. Shifting means (e.g., an actuator, such as alinear or vertical actuator, or the like) are operatively connected tothe fiber for shifting the same between its raised and loweredpositions, whereby a defined volume of liquid contained in the tube endregion is expelled from the tube onto a selected substrate disposedbelow the tube.

In a preferred embodiment, the fiber is (i) laterally flexible and (ii)substantially incompressible along its longitudinal axis. For example,the fiber can be an optical fiber. Advantageously, these characteristicspermit efficient transfer of motion from the shifting means to thefiber, and the accommodation of variations in the distance between thetube's lower end and the substrate by flexing, or bowing, of the fiber.

According to one embodiment, the fiber makes contact with the substratewhen shifted to its lowered position. In another embodiment, the fiberremains spaced apart from the substrate when shifted to its loweredposition.

The micro-spotting apparatus can be used to micro-spot a predeterminedvolume of a liquid at a preselected position at each of a plurality ofsubstrates. In one such embodiment, the apparatus further includespositioning means for positioning the tube and associated fibersuccessively at the preselected position. Fiber flexing accommodatesvariations in the distance between the tube's lower end and thedifferent substrate positions.

In another embodiment, the apparatus is adapted for use inmicro-spotting a predetermined volume of one or more selected liquidssimultaneously at multiple selected deposition regions on a substrate.In this embodiment, the apparatus further includes a plurality of tubes,and associated fibers and shifting means. In an exemplary arrangement,the tube end regions have diameters of less than about 200 μm, thefibers are flexible fibers, and the fibers in their lowered positionsare adapted to make contact with the substrate. Additionally, fiberflexing accommodates variations in the distance between the tubes' lowerends and the associated positions on the substrate.

Another aspect of the present invention provides an apparatus forproducing an array of liquid-reagent spots on a substrate.

In one embodiment, the array-producing apparatus includes a manifold, orchannel assembly, having a plurality of capillary channels, each adaptedto hold a selected liquid. The channels have opposite upper-end andlower-end openings, and inner diameters that decrease on progressingfrom the upper-to the lower-end openings. The lower-end openings, inthis embodiment, define the pattern and center-to-center spacing, orpitch, of the spot array. A support is movable between raised andlowered positions with respect to the manifold. A plurality of fibersare suspended from the support for movement therewith. Each fiber isadapted to move longitudinally within an associated channel, as thesupport is moved between its raised and lowered positions. Movement ofthe fibers from their raised to lowered positions is effective todeposit a selected volume of liquid from each channel in the manifold.

One embodiment further provides shifting means operatively connected tothe support for shifting the same between its raised and loweredpositions.

The spacing between adjacent upper-end openings of the manifold can bethe same as that between adjacent lower end openings, or it can differ.In one embodiment, the spacing between adjacent upper-end openings issubstantially greater than that between adjacent lower-end openings. Forexample, the spacing between lower-end openings can be one half, onethird, or one fourth that of the upper-end openings.

According to one embodiment, the fibers are adapted to contact anunderlying substrate, with the support in its lowered position, andvariations in the length of fiber extending between its associatedchannel end and its point of contact on the substrate is accommodated byfiber flexing.

In one particular construction, the diameter of the channels at theirlower ends is less than about 200 μm, and between about 10-100 μm largerthan that of an associated fiber.

In another exemplary construction, each channel has a substantiallyuniform diameter extending along a lower end region that terminates atthe channel's lower end. Further, the diameter of each channel endregion is substantially the same as that of the associated fiber. Thefiber's end, with such in its raised position, is disposed above thechannel end region, such that shifting of the fiber from its raised toits lowered position is effective to expel from the channel the volumeof liquid contained in the channel end region.

A further aspect of the present invention provides a valving apparatusfor use in metering a selected amount of liquid onto the surface of asubstrate.

According to one embodiment, the valving apparatus of the inventionincludes a reservoir for holding a selected liquid. A tube extends fromthe reservoir and terminates at a lower end orifice adjacent a planeadapted to be occupied by the surface of a selected substrate. A fiberis disposed in the tube for axial oscillatory movement therein, with alower portion of the fiber extending through the orifice. The innerdiameter of the tube and the diameter of the fiber are dimensioned toprevent fluid flow through the orifice in the absence of fiberoscillation. Oscillating means (e.g., an oscillating unit) operativelyconnect to the fiber for oscillating the same, including a control unitfor determining the oscillation amplitude, frequency and time applied tothe fiber, and thereby the amount of liquid allowed to pass through thetube orifice.

One embodiment of the valving apparatus further includes positioningmeans for positioning the tube and fiber with respect to the substrate,from one selected lateral position to another. In an exemplaryarrangement, the positioning means is operatively connected to the tube,fiber and oscillating means.

In one embodiment, the oscillation means is adapted to produce anoscillation frequency of at least about 10 Hertz, and preferably atleast about 100 Hertz. In another embodiment, the oscillation means isadapted to produce an oscillation amplitude of at least about 10 μm, andpreferably at least about 100 μm.

In one exemplary construction, the tube of the valving apparatus has alower-end diameter of less than about 100 μm, and the clearance betweenthe fiber and tube at its lower end is less than about 25 μm.

The fiber of the valving apparatus can remain spaced apart from theselected substrate during its oscillation cycle, or it can be adapted tocontact the substrate during at least a portion of its oscillationcycle. In one embodiment, for example, the fiber remains in contact withthe substrate throughout its oscillation cycle.

In another of its aspects, the present invention provides a method offorming a reagent spot on a substrate. According to one embodiment, themethod includes the steps of: (i) reciprocally moving an elongate,flexible fiber longitudinally within a capillary tube holding a selectedliquid at a frequency and amplitude sufficient to pump a portion of theliquid out through an orifice at a lower end of the tube, therebyforming a pendent drop; and (ii) placing the pendent drop at a selectedregion on the substrate.

According to one general embodiment, the pendent drop is placed on thesubstrate by contacting the drop and/or the tip of the fiber with theselected region of the substrate. In another embodiment, the pendentdrop is placed on the substrate by maintaining the fiber in spacedrelation over the selected region and enlarging the pendent drop untilit falls under the force of gravity.

These and other features and advantages of the present invention willbecome clear from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and manner of operation of the invention, together withthe further objects and advantages thereof, may best be understood byreference to the following description taken in conjunction with theaccompanying drawings, in which:

FIG. 1 is partially schematic, side-elevational view, with portionsshown in section, of a spotting device constructed in accordance with anembodiment of the present invention;

FIG. 2 is a partially schematic, top plan view showing components of anautomated apparatus for forming arrays in accordance with the invention;

FIGS. 3A to 3C illustrate a spotting device, and method of use, fordelivering a micro-volume of liquid onto the surface of a substrate, inaccordance with one embodiment of the present invention;

FIGS. 4A to 4C illustrate a spotting device, and method of use, fordelivering a micro-volume of liquid onto the surface of a substrate, inaccordance with a further embodiment of the present invention;

FIG. 5 illustrates a non-contact delivery method for transferring amicro-volume of liquid from a tube to the surface of a substrate using avalving apparatus as taught by the present invention;

FIG. 6 illustrates a contact delivery method for transferring amicro-volume of liquid from a tube to the surface of a substrate using avalving apparatus as taught by the present invention;

FIGS. 7A to 7E illustrate a spotting device, and method of use, fordelivering a micro-volume of liquid onto the surface of a substrate,according to an embodiment of the present invention;

FIGS. 8 and 9 are partially schematic, side-elevational views of aspotting head for laying down an array of liquid spots on the surface ofa substrate, according to an embodiment of the present invention;

FIGS. 10A to 10E illustrate an apparatus, and method of use, fordelivering a liquid reagent from a tube into the well of a microplate,as taught by the present invention; and

FIG. 11 is a partially schematic, side-elevational view of an automatedsystem for simultaneously delivering one or more liquid reagents into aplurality of wells of a microplate.

DETAILED DESCRIPTION OF THE INVENTION

The following discussion of the preferred embodiments of the presentinvention is merely exemplary in nature. Accordingly, this discussion isin no way intended to limit the scope of the invention.

One aspect of the invention provides a device for dispensing a smallvolume of a liquid reagent on a substrate. Generally, the deviceincludes a tube adapted to contain the liquid. An elongate fiber isdisposed within the tube for axial movement therein between raised andlowered positions. Upon shifting or oscillating the fiber between itsraised and lowered positions, a liquid spot can be formed at a selectedposition on the substrate.

In one exemplary arrangement of a spotting device, denoted generally as8 in FIG. 1, a fiber, indicated as 12, extends longitudinally within atube, denoted as 14, having an orifice 16 at its lower end. Tube 14 isadapted to contain a liquid reagent, such as 18, for controlleddeposition on a substrate, as discussed more fully below. Tube 14 can beformed, for example, from metal, plastic, glass, ceramic, or othermaterial(s) considered suitable by those skilled in the art. A reservoir20, disposed in fluid communication with tube 14, is adapted to receiveand hold a supply of liquid reagent. Reservoir 20 feeds liquid reagentto tube 14 as the tube's reagent content is depleted. In an exemplaryconstruction, a hypodermic needle (gauge 30, ¼ in. long, 90 deg. bluntend) is employed as the tube, and an associated plastic luer hub is usedas the reservoir.

Normally, capillary or surface tension forces prevent free flow of theliquid reagent out of the tube's lower orifice. In this regard, thetube's lower region can be of capillary size, so that capillary forcesprevent free flow of liquid reagent 18 out of orifice 16. For example,orifice 16, and a region of tube 14 extending upwardly therefrom, can beformed with an inner diameter of less than about 2 mm, and preferablyless than about 1 mm. In one particular construction, the inner diameteralong the lower region of tube 14 is less than about 200 μm. To furtherencourage the desired capillary action, the capillary-size region oftube 14 can be provided with an interior surface that is hydrophilic,i.e., wettable. For example, the interior surface of tube 14 can beformed of a hydrophilic material and/or treated to exhibit hydrophiliccharacteristics. In one embodiment, the interior surface has native,bound or covalently attached charged groups. One suitable surface is aglass surface having an absorbed layer of a polycationic polymer, suchas poly-l-lysine.

Tube 14 can be provided with an exterior surface that is hydrophobic,i.e., one that causes aqueous medium deposited on the surface to bead.For example, the exterior surface of tube 14 can be formed of ahydrophobic material and/or treated to exhibit hydrophobiccharacteristics. This can be useful, for example, to prevent spreadingof a drop, formed at the region of orifice 16, along the outer surfaceof the tube. It can also be useful to maintain a drop of liquid in theform of a globule at the lowermost tip. A variety of known hydrophobicpolymers, such as polystyrene, polypropylene, and/or polyethylene, canbe utilized to obtain the desired hydrophobic properties. In addition,or as an alternative, a variety of lubricants or other conventionalhydrophobic films can be applied to the tube's exterior surface,particularly along the tube's lower region proximate outlet 16.

With continuing reference to the embodiment of FIG. 1, the diameter offiber 12 is between about 10 to 500 μm smaller than the inner diameterof tube 14. In this regard, fiber 12 can have a diameter of betweenabout 25 to 1,000 μm. According to one embodiment, fiber 12 has adiameter of less than about 200 μm, and preferably less than about 100μm. In one particular arrangement, a fiber having a diameter of about 75μm extends longitudinally within a tube having an inner diameter ofabout 100 μm along its lower region, and an outer diameter of about 200μm. In another arrangement, a fiber having a diameter of about 50 μmextends longitudinally within a tube having an inner diameter of about75 μm along its lower region, and an outer diameter of about 200 μm.

For reasons that will become apparent, fiber 12 is preferablyconstructed to be laterally flexible and longitudinally incompressible.Materials suitable for forming fiber 12 include those typically employedin the construction of optical fibers, such as glass, plastic, silica,quartz, and the like. Suitable optical fibers are available from anumber of commercial sources. One particularly preferred fiber, having adiameter of about 0.002 inches (i.e., about 50.8 μm), is that suppliedby Edmund Scientific Co. (Barrington,. N.J.) under catalog # F54014.

As previously indicated, fiber 12 is disposed within tube 14 for axialmovement therein between raised and lowered positions. In the presentembodiment, when in the raised position, the fiber's free end or tip 12a is spaced apart from the surface of a selected substrate, such asslide 22. When shifted to the lowered position, tip 12 a approaches thesurface of substrate 22.

Shifting means are operatively connected to fiber 12 for shifting thesame between its raised and lowered positions. The shifting means caninclude, for example, an actuator that is movable between two positions,such as a linear or vertical actuator, or the like. In the embodiment ofFIG. 1, for example, a solenoid assembly 24 is activatable to draw asolenoid piston 26 downwardly, then release the piston, e.g., underspring bias, to a normal, raised position (shown in dashed lines). Manysolenoids are available from commercial sources, and suitable models canbe readily chosen by those skilled in the art. One particular solenoid,contemplated for use herein, is available from Jameco ElectronicsComponents (Belmont, Calif.) under part # 145314 (12 VDC ContinuousTubular Push/Pull Solenoid). In one embodiment, the solenoid is operableto shift the fiber up and down over a stroke of about 1 mm.

Other actuators, useful for shifting the fiber, include, for example,pneumatic, hydraulic, magnetostrictive, and piezoelectric actuators, aswell as motor assemblies (e.g., steppers) operable to generate adownward motive force followed by reciprocation. Several particularassemblies which can be readily adapted for use herein as the shiftingmeans are disclosed, for example, in U.S. Pat. Nos. 3,164,304;3,329,964; 3,334,354; 5,443,791; 5,525,515; 5,551,487; 5,601,980; and5,807,522; each of which is expressly incorporated herein by reference.

Positioning means can be utilized to move the spotting device linearlyor in an x-y plane to position the spotting device at a selecteddeposition position. In this regard, certain embodiments provide formovement of the tube while the target substrate is held stationary.According to other embodiments, the tube remains in a fixed position,while the substrate is shuttled into position. Still further embodimentsprovide for movement of both the tube and the target substrate, eithersequentially or in parallel.

In one exemplary arrangement of the positioning means, the spottingdevice is carried on an arm of an x-y positioner. The arm, in thisarrangement, can be moved either linearly or along an x-y plane toposition the spotting device at a selected deposition position. Suchmovement can be accomplished, for example, using a robotic assembly, orthe like. Exemplary robotic devices include, for example, robots withelectronically controlled linked or crossed movable arms, such as aSCARA, gantry and Cartesian robots. It is understood, of course, thatany other robotic mechanism could be used in accordance with the presentinvention so long as it can accomplish substantially the same purposesand secure substantially the same result. In this regard, cylindricalrobots, polar robots, articulated robots, or the like can be utilized.In one embodiment, the positioning means comprises a motorized x-ycarriage or rail assembly. For example, an AUTOMOVE® 402, available fromAsymtek (Carlsbad, Calif.), can be used for x-y positioning and solenoidactivation.

The arm that supports the tube, in the above arrangement, can include aclamp capable of releasably grasping the tube. This arrangement permitsswapping of the tube with a different one, e.g., loaded with a differentliquid reagent, as desired.

One particular positioning means, contemplated for use herein, will nowbe described in detail. The apparatus is shown in planar, and partiallyschematic view in FIG. 2. A spotting device 8 in the apparatus has thebasic construction described above with respect to FIG. 1, and includesa tube 14 terminating at a lower orifice. A fiber 12, disposed axiallywithin the tube 14 of the spotting device 8, is adapted for movementtoward and away from the surface of a substrate, to dispense a selectedvolume of liquid sample or reagent, as described herein. A solenoid 24,or other shifting means, effects this movement. Solenoid 24 is under thecontrol of a control unit 52 whose operation will be described below.

The spotting device is carried on an arm 54 that is threadedly mountedon a worm screw 58 driven (rotated) in a desired direction by a steppermotor 62 also under the control of unit 52. At its left end in thefigure, screw 58 is carried in a sleeve 64 for rotation about the screwaxis. At its other end, the screw is mounted to the drive shaft of thestepper motor, which in turn is carried on a sleeve 66. The spottingdevice, worm screw, the two sleeves mounting the worm screw, and thestepper motor used in moving the device in the “x” (horizontal)direction in the figure form what is referred to here collectively as adisplacement assembly 66.

The displacement assembly is constructed to produce precise, micro-rangemovement in the direction of the screw, i.e., along an x axis in thefigure. In one mode, the assembly-functions to move the spotting devicein x-axis increments having a selected distance in the range 5-500 μm.In another mode, the spotting device may be moved in precise x-axisincrements of several microns or more, for positioning the spottingdevice at associated positions on adjacent substrates, as will bedescribed below.

The displacement assembly, in turn, is mounted for movement in the “y”(vertical) axis of the figure, for positioning the spotting device at aselected y axis position. The structure mounting the assembly includes afixed rod 68 mounted rigidly between a pair of frame bars 70, 72, and aworm screw 74 mounted for rotation between a pair of frame bars 76, 78.The worm screw is driven (rotated) by a stepper motor 80 that operatesunder the control of unit 52. The motor is mounted on bar 76, as shown.

The structure just described, including worm screw 74 and motor 80, isconstructed to produce precise, micro-range movement in the direction ofthe screw, i.e., along a y axis in the figure. As above, the structurefunctions in one mode to move the spotting head in y-axis incrementshaving a selected distance in the range 5-500 μm, and in a second mode,to move the spotting head in precise y-axis increments of severalmicrons or more, for positioning the spotting head at associatedpositions on adjacent substrates.

A workpiece holder 82 in the apparatus functions to hold a plurality ofsubstrates, such as substrates 22 on which the microarrays of reagentregions are to be formed by the apparatus. The holder provides a numberof recessed slots, such as slot 86, which receive the substrates, andposition them at precise selected positions with respect to the framebars on which the spotting device positioning means is mounted.

As noted above, the control unit in the device functions to actuate thetwo stepper motors and solenoid in a sequence designed for automatedoperation of the apparatus in forming a selected microarray of reagentregions on each of a plurality of substrates.

The control unit is constructed, according to conventionalmicroprocessor control principles, to provide appropriate signals to thesolenoid and each of the stepper motors, in a given timed sequence andfor an appropriate signaling time. The construction of the unit, and thesettings that are selected by the user to achieve a desired arraypattern, will be understood from the following description of a typicalapparatus operation.

Initially, one or more substrates are placed in one or more slots in theholder. Motors 62, 80 are then actuated to position the spotting deviceat a selected array position at the first of the substrates. Solenoidactuation of the spotting device is then effected to dispense aselected-volume aliquot of that reagent at this location. This operationis effective, for example to dispense a selected volume preferably lessthan about 1 μl (e.g., between about 2 pl and 2 nl) of the liquidreagent.

The spotting device is now moved to the corresponding position at anadjacent substrate and a similar volume of the liquid reagent isdispensed at this position. The process is repeated until the reagenthas been dispensed at this preselected corresponding position on each ofthe substrates.

Where it is desired to dispense a single reagent at more than two arraypositions on a substrate, the spotting device may be moved to differentarray positions at each substrate, before moving the spotting device toa new substrate, or liquid reagent can be dispensed at individualpositions on each substrate, at one selected position, then the cyclerepeated for each new array position.

To dispense the next reagent, the spotting device is exchanged foranother such device containing a different selected reagent. The processof dispensing the reagent at each of the corresponding second-arraypositions is then carried out as above. This process is repeated untilan entire microarray of liquid reagents on each of the substrates hasbeen formed.

Several other x-y positioning assemblies which can be readily adaptedfor use herein as the positioning means are disclosed, for example, inU.S. Pat. Nos. 5,443,791; 5,551,487; and 5,587,522; each of which isexpressly incorporated herein by reference.

As previously mentioned, the positioning means can instead, or inaddition, be adapted to move the substrate to a spotting position. Inthis regard, the substrate can be adapted for manipulation by a roboticassembly, or it can be supported on a conveyor, or an x-y movable stageor platform.

Any desired substrate(s) can be used with the present invention,including slides, cards, plates, chips, and the like. In one generalembodiment, the substrate surface is relatively hydrophilic, i.e.,wettable. For example, the surface can have native, bound or covalentlyattached charged groups. One such surface is a glass surface having anabsorbed layer of a polycationic polymer, such as poly-l-lysine. In oneembodiment, for example, an aqueous or predominantly aqueous reagentsolution or biological sample is spotted onto a slide having ahydrophilic surface. In another embodiment, the substrate surface has oris formed to have a relatively hydrophobic character, i.e., one thatcauses aqueous medium deposited on the surface to bead. A variety ofknown hydrophobic polymers, such as polystyrene, polypropylene, orpolyethylene have desired hydrophobic properties, as do a variety oflubricant or other hydrophobic films that may be applied to thesubstrate surface.

In some cases, it is desired to spot out the reagents in a humidenvironment so that the droplets do not dry until the arraying operationis complete.

Several exemplary devices and methods for spotting a substrate aredepicted in FIGS. 3 to 6. Generally, a spotting device of the inventionis positioned over a selected region of a substrate. In a typicaloperation, this is accomplished by aligning the selected region of thesubstrate with the spotting device such that the selected regionintersects a line defined by an extrapolation of the spotting device'scentral longitudinal axis. An elongated fiber adapted for movementaxially within the spotting device is then shifted or oscillated betweenits raised and lowered positions in a manner effective to transfer analiquot of liquid from the tube onto the selected region of thesubstrate. It should be noted that any of the above shifting means andpositioning means can be used in connection with the followingembodiments. Also, any suitable control unit, such as 52 in FIG. 2, canbe employed.

It should also be noted that, as the fiber reciprocates in a cycle fromits raised position to its lowered position and back, the fiber'sterminal end or tip will travel through a path referred to herein as the“stroke.” Along its forward stroke, the tip travels from a raised peakto a lowered peak. Along its back stroke, the tip travels from itslowered peak back to its raised peak.

In one embodiment, shown in FIGS. 3A to 3C, at the beginning of itsstroke, i.e., at its raised peak, the tip 12 a of fiber 12 is situatedoutside of tube 14, such that a section of fiber 12 intersects a planedefined by the terminal rim of tube 14 at orifice 16. From thisbeginning position, illustrated in FIG. 3A, the fiber's tip 12 a ismoved toward the surface of a selected substrate, such as slide 22,eventually reaching its lowered peak whereat tip 12 a contacts aselected substrate region 22 a, as shown in FIG. 3B. It should be notedthat fiber 12 carries with it a layer of liquid reagent 18 on itsexterior surface, including at the surface of tip 12 a. Upon contactingthe substrate, a small and controlled portion of liquid reagent isdelivered from tip 12 a to the selected region 22 a of substrate 22.After contacting substrate 22, fiber 12 is shifted back to its raisedposition, leaving behind a spot of liquid, as at 28, on the substrate'ssurface. The spotting device can then be positioned over anotherselected region to lay down an additional spot, if desired. Therepetition rate employed can be a few strokes per second, e.g., withinthe range of about 1-10 Hz. In one embodiment, the repetition rate isabout 5 strokes per second.

It should be appreciated that an exacting tolerance between the spottingdevice and substrate is not critical to achieve successful results usingthe spotting device of the present invention. That is, the fiber canretreat back to its raised position after only barely touching thesubstrate surface, or the fiber can be lowered farther than what isnecessary toward the substrate surface in order to bring its tip intocontact therewith. Advantageously, the flexibility of the fiber permitsthe fiber to flex, or bow, once the tip abuts the substrate surface.That is, flexing of the fiber can accommodate variations in the distancebetween the lower end of the spotting device and the surface of thesubstrate. This is shown in exaggerated fashion in FIG. 3B forconvenience of illustration.

It should also be appreciated that the longitudinal incompressibility ofthe fiber provides for the efficient transmission of motion from theshifting means to the fiber's tip. Advantageously, this property permitsthe use of fibers of varying lengths, including relatively long fibers(e.g., 10, 20, 30 cm, or more).

In another embodiment (not shown), similar to the embodiment justdescribed, the forward stroke can bring the fiber's tip very close tothe selected surface region of the substrate, without actually makingcontact. This permits liquid reagent on the fiber's terminal end totouch and adhere to the substrate surface, while actual physical contactbetween the tip itself and the substrate surface is avoided. Whileadvantageous for certain purposes, it will be appreciated that thisembodiment will generally be less desirable than the previouslydescribed embodiment, since an exacting tolerance between the spottingdevice and the substrate surface is required in this case.

Another spotting method is depicted in FIGS. 4A to 4C. Here, at thebeginning of its stroke, i.e., at its raised peak, the tip 12 a of fiber12 is situated within tube 14, above the plane defined by the terminalrim of tube 14 at orifice 16. From this beginning position, illustratedin FIG. 4A, the fiber tip 12 a is moved toward the surface of a selectedsubstrate, such as slide 22. Plunger- or piston-like action of tip 12 aacts to push an aliquot of liquid reagent 18 from the lower end regionof tube 14. The amount of liquid pushed out in this manner will depend,in part, on the volume of liquid occupying the region under tip 12 a atthe time tip 12 a is moved toward substrate 22. Thus, the location ofthe tip's raised peak within the tube, in this embodiment, willtypically be determined, at least in part, by the quantity of liquidthat one desires to spot on a substrate. Other variables that can beadjusted in order to control the amount of liquid deposited in thisembodiment include the surface area of tip 12 a, and the distance of thegap separating the fiber from the tube's interior surface.

As tip 12 a is moved toward substrate through its stroke, it eventuallyreaches its lowered peak, in contact with substrate 22. After contactingsubstrate 22, fiber 12 is shifted back to its raised position, leavingbehind a spot of liquid, as at 28 in FIG. 4C, on the substrate'ssurface. The spotting device can then be positioned over anotherselected region to lay down an additional spot, if desired. As with thepreviously described embodiment, the repetition rate employed can be afew strokes per second, e.g., within the range of about 1-10 Hz. In oneembodiment, the repetition rate is about 5 strokes per second.

It should be appreciated that the piston-like action of fiber 12, asjust described, causes a thicker layer of liquid to form at the fiber'slower end region, as compared to the embodiment of FIGS. 3A to 3C. Thus,it is contemplated that this embodiment will be used to create reagentspots having a greater volume.

In another embodiment (not shown), similar to the embodiment justdescribed, the fiber's tip can be moved toward the selected surfaceregion of the substrate, without making contact. For example, movementof the fiber tip toward the substrate can be abruptly stopped, orreversed, so that liquid is thrown or ejected, e.g., as by inertia, fromthe fiber onto the substrate. Or, the fiber tip can be moved into veryclose proximity to the substrate surface, without physically contactingit, so that liquid reagent carried on the fiber touches and adheres tothe substrate.

A further aspect of the present invention provides a valving apparatusfor use in metering a selected amount of liquid onto the surface of asubstrate. In one embodiment, shown in FIGS. 5 and 6, a tube 14communicates at its upper end with a reagent-supply reservoir 20, andterminates at a lower end orifice 16, much like the previously describedspotting devices. A fiber 12 is disposed in tube 14 for axialoscillatory movement therein.

The inner diameter of tube 14 and the diameter of fiber 12 aredimensioned to prevent fluid flow through orifice 16 in the absence offiber oscillation. In one embodiment, for example, tube 14 has alower-end diameter of less than about 200 μm, and preferably less thanabout 100 μm, and the clearance between the fiber and tube at its lowerend is less than about 50 μm, and preferably less than about 25 μm.

Oscillating means operatively connect to fiber 12 for oscillating it.The oscillating means can comprise, or example, an oscillating unithaving a control unit for determining the oscillation amplitude,frequency and time applied to the fiber, and thereby the amount ofliquid allowed to pass through orifice 16. The oscillating unit cancomprise any device capable of oscillating the fiber axially within thetube in such a controlled manner. Suitable oscillating units caninclude, for example, a solenoid or motor assembly, or a pneumatic,hydraulic, magnetostrictive, or piezoelectric actuator. In oneembodiment, the oscillating means is adapted to produce an oscillationfrequency of at least about 10 Hz, and preferably at least about 100 Hz.Preferably, the oscillating means is adapted to produce an oscillationamplitude of at least about 10 μm, and preferably at least about 100 μm.

The valving apparatus can further include positioning means forpositioning the tube and fiber with respect to the substrate, from oneselected lateral position to another. In one embodiment, the positioningmeans is operatively connected to the tube, fiber and oscillating means.The positioning means can comprise devices as set forth above withregard to the spotting device.

In operation, fiber 12 can be moved axially within the liquid-holdingtube in a reciprocal fashion. Such oscillatory movement occurs at afrequency and amplitude, and for a length of time, sufficient to pump aselected quantity of liquid reagent 18 out through orifice 16 at a lowerend of tube 14, thereby forming a pendent drop, such as at 18 a in FIG.5. The pendent drop can be placed at a selected region of a substrate bycontacting the drop 18 a with the selected substrate region. In oneembodiment, the fiber's tip 12 a is moved into contact with thesubstrate. Such contact can be periodic, e.g., once per oscillation ofthe fiber, or it can be continuous such that the fiber engages thesubstrate throughout its oscillation cycle. It should be appreciatedthat formation of such a “liquid bridge,” as illustrated in FIG. 6,permits the creation of relatively large reagent spots. Alternatively,the pendent drop can be placed on the substrate by maintaining the fiberin spaced relation over the selected region and enlarging the pendentdrop until it falls under the force of gravity.

Another exemplary spotting device of the present invention is depictedin FIGS. 7A to 7E. Similar to the previous embodiments, a flexible fiber12 extends longitudinally through a tube 14 having a lower orifice 16.In this embodiment, however, the inner diameter of tube 14 tapers onprogressing downwardly to a defined-volume tube end region, indicatedgenerally at 14 a, having a substantially uniform diameter. Preferably,the tube end region 14 a diameter is substantially the same as thediameter at orifice 16. Further, the inner diameter along the lower endregion 14 a of tube 14, proximate orifice 16, is very close to (e.g.,within about 10 μm), and preferably substantially the same as, thediameter of fiber 12. For example, in one embodiment, both the fiber'sdiameter and the inner diameter of the tube, along region 14 a, are lessthan about 200 μm; and are preferably about 100 μm. In anotherembodiment, both of these values are about 50 μm. The upper, largerdiameter portion of the tube can act as a supply reservoir 20 forfeeding liquid reagent to the lower tube portion as spotting operationsare effected.

Shifting means are operatively connected to fiber 12 for shifting thefiber between its raised and lowered positions. The apparatus canfurther include positioning means for positioning the tube and fiberwith respect to the substrate, from one selected lateral position toanother. In one embodiment, the positioning means operatively connect tothe tube, fiber and oscillating means. The shifting means and thepositioning means can comprise devices as previously set forth herein.

Steps of a typical operation are depicted sequentially in FIGS. 7Athrough 7E. Initially, the shifting means (not shown) shifts fiber 12from its raised position, of FIG. 7A, towards a substrate, such as slide22. Notably, when fiber 12 is in its raised position, its tip 12 a isdisposed above the tube end region 14 a. As fiber 12 is shifted from itsraised to its lowered position, the volume of liquid reagent 18occupying the tube end region 14 a is expelled from tube 14, as depictedin FIG. 7B. When fiber 12 reaches its lowered position, as shown in FIG.7C, the expelled liquid reagent is transferred to a selected region 22 aof substrate 22. As fiber 12 is shifted back to its raised position, asshown in FIGS. 7D and 7E, a spot of liquid reagent 28 is left behind onthe surface of substrate 22. When the fiber is fully retracted back intothe fluid container (FIG. 7E), another deposition cycle is ready tobegin, if desired.

As with those previous embodiments involving contact between the fiberand the substrate, it should be appreciated that an exacting tolerancebetween the spotting device and substrate is not critical to achievesuccessful results using the just-described spotting device.Advantageously, the flexibility of the fiber permits the fiber to flex,or bow, once the tip abuts the substrate surface, as shown inexaggerated fashion in FIG. 7C for convenience of illustration. That is,flexing of the fiber can accommodate variations in the distance betweenthe lower end of the spotting device and the surface of the substrate.It should also be appreciated that the longitudinal incompressibility ofthe fiber provides for the efficient transmission of motion from theshifting means to the fiber's tip.

One embodiment provides an operation substantially like that of FIGS. 7Ato 7E, except that the fiber's lower tip never makes contact with thesubstrate surface. For example, movement of the fiber tip toward thesubstrate can be abruptly stopped, or reversed, so that liquid is thrownor ejected, e.g., as by inertia, from the fiber onto the substrate. Or,the fiber tip can be moved into very close proximity to the substratesurface, without physically contacting it, so that liquid reagentexpelled by the fiber touches and adheres to the substrate.

Still a further aspect of the present invention provides a hand-operablespotting device (not shown). The tube, in this embodiment, isdimensioned to fit comfortably in the hand of an operator. In thisregard, the outer dimensions of the hand device are preferably similarto those of typical writing implements, such as ink pens, mechanicalpencils, and the like. For example, the tube can have an outer diameterof between about 0.75-1.50 cm and a length of between about 10-20 cm. Anelongate, flexible fiber, e.g., an optical fiber, is disposed within thetube for axial movement therein between raised and lowered positions.The tube is adapted to hold, e.g., by way of capillary forces, aselected liquid reagent for deposition. The interior dimensions of thetube, and the dimensions of the fiber, can be as set out for any of theforegoing embodiments.

The fiber of the hand device attaches at its upper end to a piston thatis adapted for reciprocal movement within the tube. Normally, the pistonis urged away from a lower orifice of the tube, toward the top of thetube, by a coil spring or other biasing mechanism. At this position, thefiber is shifted toward its raised position, substantially retractedinto the tube. The shifting means, in this embodiment, includes adepressible shaft having an end region that protrudes through an openingat the upper end of the tube. The other end of the shaft, located withinthe tube, operatively engages the piston. Upon depressing the rod, e.g.,using the thumb of an operator's grasping hand, the normal biasing forceof the coil spring can be overcome, so that the piston is pusheddownwardly through the tube. Such movement of the piston causes thefiber to move axially within the tube, so that the fiber's lower endprotrudes from the tube's lower orifice. At this position, a liquid spotcan be transferred from the fiber's tip to a selected position on asubstrate. Upon releasing the depressible shaft, the coil spring returnsthe fiber to its raised position.

In another of its aspects, the present invention provides a spottinghead for producing an array of liquid-reagent spots on a substrate. Asdiscussed more fully below, the spotting head of the invention can beadapted to lay a great number, e.g., hundreds or thousands, of spots persecond.

One embodiment of the spotting head is shown in FIG. 8. In thisembodiment, a conduit or channel assembly 101, also referred to hereinas a manifold, includes a plurality of channels, such as 114 a-114 b.Each channel 114 a, 114 b has opposite upper-end and lower-end openings,as at 115 a-115 b and 116 a-116 b, respectively. The channels aremaintained in fixed, spaced relation to one another. In one embodiment,the channels take the form of tubes, barrels or funnels that are secured(e.g., snap fit) into a frame or rack body. In another embodiment, thechannels are of a monolithic construction. For example, the channelassembly can be integrally constructed of plastic using an injectionmolding process; or each channel can be formed by boring through a blockof material, such as glass, plastic, metal, or the like.

The inner diameter of each channel decreases on progressing from theupper- to the lower-end openings. For example, the channels can begenerally cone-shaped or horn-shaped channels. The longitudinal axis ofeach channel can be straight, angled, curved, or other suitable shape.In this regard, attention is directed to the generally S-shaped channelsshown in FIG. 8.

A region of each channel extending from a respective one of thelower-end openings is of capillary size, such that a liquid, e.g., abiological sample or reagent solution, placed in the channel willnormally be maintained therein by way of capillary forces. Any innerdiameter that effects the desired capillary action can be utilizedwithin the scope of this invention. For example, the capillary-sizeregions can be formed with an inner diameter of less than about 1 mm,and preferably less than about 200 μm. To further encourage the desiredcapillary action, the capillary-size region of each conduit can beprovided with an interior surface that is hydrophilic.

While only six channels, arranged side-by-side in a linear fashion, arevisible in the view of FIG. 8, it should be understood that anyreasonable number of channels can be disposed in any desired spatialconfiguration. For example, the manifold can include 24, 48, 96, 384,1024, 1536 channels, or more. In such arrangements, the channel upper-and lower-end openings will typically be arranged in a regular array,e.g., an 8×12, 16×24, 32×32 or a 32×48 array, though other layouts arepossible.

A support, denoted as 105, is adapted for movement between raised andlowered positions with respect to manifold 101. In the embodiment ofFIG. 8, this is accomplished by providing a frame, such as 107, having atrack comprised of spaced-apart, parallel linear rail portions, denotedas 109 a and 109 b, along which support 105 is guided. For example,support 105 can have a groove or slot (not shown) formed along each ofits side-end regions, proximate rails 109 a, 109 b, with each slot beingslidably mounted over a respective one of the rails.

Movement and positioning of support 105 along the track can be effectedby way of manual or automatic shifting means. In this regard, and withadditional reference to FIG. 9, a motor assembly 121 communicates with acontroller 123 and power supply 125. A flexible wire or line 127 extendsbetween motor 121 and support 105. One end of wire 127 is connected toan upper side of support 105. The other end of wire 127 is secured to aspool (not shown) which, in turn, is adapted for rotation by motorassembly 121. In one mode, motor assembly 121 can rotate the spool so asto wind wire 127 therearound, thereby moving support 105 up along thetrack towards its raised position (FIG. 8). In another mode, motorassembly 121 can rotate the spool so as to unwind, and thus extend, wire127 therefrom, thereby moving support 105 down the track towards itslowered position (FIG. 9). Rather than employing a wire to communicatethe motor with the support, other embodiments contemplate the use ofgear assemblies. It should be noted that other automatic shifting means,suitable for use herein, include, for example, hydraulic or pneumaticactuators. Alternatively, support 105 can be shifted by hand.

A plurality of fibers, such as optical fibers 112 a-112 b, are carriedon support 105 for movement therewith. The fibers can be secured to thesupport in any suitable manner. For example, the upper end region ofeach fiber can be received within a respective bore extending up fromthe lower side of support 105. Each fiber's upper end can be held in itsbore, for example, by way of frictional forces and/or by usingconventional adhesives. The fibers are arranged such that the spacingbetween adjacent fibers substantially matches the spacing betweenadjacent upper-end openings of manifold 101, allowing insertion of onefiber per channel as support 105 is moved towards its lowered position.In a typical arrangement, the fibers will be disposed in a regulararray.

The diameter of the fibers, extending from support 105, as well as theinterior dimensions of the capillary-size regions of the channels, canbe like that set out for any of the previous embodiments. In anexemplary arrangement, the diameter of each channel is less than about200 μm, and each channel is between about 10-100 μm larger than that ofan associated fiber. In another arrangement, the interior diameter alongthe lower end region of each channel is substantially the same as thatof an associated fiber.

Upon moving support 105 to its lowered position, the lower tip of eachfiber is passed through a respective channel of manifold 101 and broughtto a plane adapted to be occupied by a substrate. As shown in FIG. 9,when a substrate, such as plate 22, occupies such plane, the fiber tipsabut the substrate's surface at this position. In this way, each fibercan transfer an aliquot of a liquid reagent held in its respectivechannel to the surface of the substrate. It should be noted that thelower-end opening array of manifold 101 defines the array of spotsformed on substrate 22.

As best viewed in FIG. 8, the fibers extending from support 105 are notall of the same length. Rather, they are cut such that upon beinglowered through their respective channels they will contact anunderlying substrate at roughly the same time, or within a short time ofone another. Appropriate fiber lengths can be established by passing thefibers through the channels, and then cutting each fiber at its regionintersecting the plane adapted to be occupied by the surface of asubstrate. It is not critical to the successful operation of theinvention that the various fibers be cut with exacting precisionrespective to the substrate plane, since variations in the length offiber extending between its associated channel end and its point ofcontact on the substrate can be accommodated by fiber flexing.

For applications requiring formation of an array of reagent spots havingthe same center-to-center spacing, or pitch, between adjacent spots asbetween adjacent fibers of the fiber array, the pitch of both the upper-and lower-opening arrays can be made about equal. For example, in oneembodiment, each of (i) the fiber array, (ii) the upper-end openingarray, and (iii) the lower-end opening array all have substantially thesame center-to-center spacing. For applications requiring the formationof a reagent spot array having a reduced pitch as compared to the fiberarray, on the other hand, it will be advantageous to utilize a channelassembly having upper- and lower-end opening arrays that differ inpitch, as well. In one exemplary arrangement of this type, the channellower-end opening array is provided with a center-to-center pitch thatis smaller than that of the upper-end opening array. FIGS. 8 and 9 show,for example, an embodiment wherein the pitch of the lower-end openingarray is substantially smaller than that of the upper-end opening array.For example, the center-to-center pitch of the lower-end opening arraycan be between about ½ to ¼ that of the upper-end opening array. In oneparticular embodiment, the center-to-center spacing of the lower-endopening array is about ⅓ that of the upper-end opening array.

According to one embodiment, a plurality of substrates are sequentiallyshuttled under the spotting head. For example, a conveyor can carry anumber of tandemly-arranged substrates along a transport pathway passingunder a manifold. The channels of the manifold can hold a single type ofliquid reagent, or multiple types of liquid reagents. Upon positioning aselected substrate beneath the manifold, the conveyor can pause. At thispoint, the fibers can be shifted between their raised and loweredpositions to lay down an array of spots on the substrate. If desired,such shifting can be repeated one or more times to transfer additionalliquid to the substrate. Such additional liquid can be placed at thealready-laid spots, or, upon incrementally moving the substratelaterally under the manifold, at previously unspotted regions of thesubstrate. The just-spotted substrate can then be moved out from underthe manifold and a new, upstream substrate can be moved into positionfor spotting. If desired, several spotting heads can be situated atrespective positions along the transport pathway. In one embodiment, aconveyor shuttles one or more substrates along a transport pathwayextending under several spotting heads that are disposed sequentiallyalong the transport pathway, at positions that are laterally offset fromone another. This arrangement can provide a very compact interleaving ofspots on a given substrate, even though the spots laid by any onespotting head have a wider spacing.

Changeover from one set of liquid reagents to a different set, orreplacement of an emptied manifold with a loaded one, can beaccomplished in a quick and efficient manner. For example, an operatoror robot can simply remove the present manifold and insert another inits place. In this regard, the manifold can be configured to removablysnap-lock into the frame. Where one manifold holding a first set ofliquid reagents is swapped for another manifold holding a second,different set of reagents, the fiber array can be readily changed aswell. To this end, the fiber support can also be constructed toremovably snap-fit into the frame.

Refilling low or emptied channels of the manifold can also be readilyaccomplished. In this regard, the upper-end openings of the channels canbe dimensioned large enough (e.g., >3 mm diameter) to permit readyaccess to conventional means of fluid loading, such as pipettes orsyringes.

In one embodiment, both the manifold and the fiber support areconstructed of relatively inexpensive materials, e.g., plastics, metalor glass, using conventional tooling and/or molding procedures. Bykeeping the component cost low, it can be cost-effective to throw awaythe fiber support (with the fibers) and/or the manifold when a newliquid reagent set is introduced. Disposing of one or both of thesecomponents, rather than cleaning and re-using them, eliminates apotential source of contamination. Furthermore, utilization ofdisposable components helps avoid the time, equipment, and labor costsassociated with cleaning/drying efforts. In contrast, most conventionalspotting systems, such as quill, ink-jet, or pin, must be cleaned eachtime a new fluid is deposited.

The above-described spotting devices and methods provide a relativelylow-energy approach to liquid deposition. For example, the spotting headcan employ a highly parallel approach to lay many spots (e.g., hundredsor thousands) per second. Notably, the process of laying any single spotis a relatively slow process. For example, each individual fiber mightlay only one or a few spots per second. Consequently, problemsassociated with very energetic spot deposition such as splattering andmisdirected ejection (satellites), and contamination resultingtherefrom, are avoided by the present invention.

The spotting devices described herein offer reduced reagent loss ascompared to most conventional deposition systems. According to thepresent invention, liquids that are deposited onto a substrate aredirectly transferred from a tube or channel onto the surface of asubstrate without the use intermediate containers. It should beappreciated that intermediate containers typically waste fluid becauseof residues and films that are unavoidably left behind. For applicationsrequiring very small amounts of fluid (e.g., a micro-liter or less),intermediate containers such as reservoirs in ink-jets or the split in aquill can waste an unacceptable amount of fluid.

Still a further aspect of the present invention provides a method andapparatus for dispensing a liquid reagent into a well or depressionformed, for example, in a tray or plate. With reference to FIGS. 10A to10E, a protrusion, such as a spike 217, extends upwardly from the bottomof a well 219 of a multi-well tray 222. A liquid container, such as anelongate tube 216, holds a liquid 218 for dispensing. Tube 216 isadapted to hold the liquid by capillary or surface tension forces. Asshown in FIGS. 10A to 10B, a meniscus 218 a can form at the lower regionof tube 216. Liquid 218 is dispensed by shifting tube 216 toward well219 until spike 217 pierces meniscus 218 a. The spike, which preferablyhas a wettable surface, draws liquid from the tube into the well.

FIG. 11 shows an automated system for simultaneously delivering one ormore liquid reagents into a plurality of wells of a plate or tray. Thesystem includes a manifold or channel assembly 301, similar to thatdescribed above with respect to FIGS. 8 and 9, adapted for shiftingbetween raised and lowered positions over a substrate, such asmulti-well tray 322. Manifold 301 includes a plurality of channels, suchas 314 a and 314 b, each having a lower-end region substantially likethe interior of tube 216 of FIGS. 10A to 10E. Shifting means areoperable to shift manifold 301 between its raised and lowered positions.In the embodiment of FIG. 11, for example, a support 305 is configuredto releasably engage an upper region of manifold 301. Support 305, inturn, is adapted to ride along a pair of parallel rails 309 a, 309 bprovided on a frame assembly 307. A motor 321, controller 323, and powersupply 325 are operable to move support 305, and thus manifold 301, upand down along rails 309 a, 309 b via wire 327. Although not visible inFIG. 11, each well of tray 322 includes a protrusion, such as a spike,extending upwardly from its floor. The spikes are adapted to extractliquid reagent from respective channels when manifold 301 is shiftedtowards its lowered position in a fashion substantially as shown inFIGS. 10A-10E.

Those skilled in the art can now appreciate from the foregoingdescription that the broad teachings of the present invention can beimplemented in a variety of forms. Therefore, while this invention hasbeen described in connection with particular embodiments and examplesthereof, the true scope of the invention should not be so limited.Various changes and modification may be made without departing from thescope of the invention, as defined by the appended claims.

It is claimed:
 1. A method of forming a reagent spot on a substrate,comprising: reciprocally moving an elongate, flexible fiberlongitudinally within a capillary tube holding a selected liquid at afrequency and amplitude sufficient to pump a portion of the liquid outthrough an orifice at a lower end of the tube, thereby forming a pendentdrop; and placing the pendent drop at a selected region on thesubstrate.
 2. The method of claim 1, wherein the pendent drop is placedon the substrate by contacting an end of the fiber with the selectedregion of the substrate.
 3. The method of claim 1, wherein the pendentdrop is placed on the substrate by maintaining the fiber in spacedrelation over the selected region and enlarging the pendent drop untilit falls under the force of gravity.